Methods of testing for agonists and antagonists of cell surface receptors

ABSTRACT

Disclosed is a transformed yeast cell containing a first heterologous DNA sequence which codes for a mammalian G protein coupled receptor and a second heterologous DNA sequence which codes for a mammalian G protein a subunit (mammalian G α ). The first and second heterologous DNA sequences are capable of expression in the cell, but the cell is incapable of expressing an endogenous G protein α-subunit (yeast G α ). The cells are useful for screening compounds which affect the rate of dissociation of G α  from G βτ  in a cell.  
     Also disclosed is a novel DNA expression vector useful for making cells as described above. The vector contains a first segment comprising at least a fragment of the extreme amino-terminal coding sequence of a yeast G protein coupled receptor. A second segment is positioned downstream from the first segment (and in correct reading frame therewith), with the second segment comprising a DNA sequence encoding a heterologous G protein coupled receptor.

RELATED INFORMATION

[0001] This application is a continuation of application Ser. No. 08/623,284 filed on Mar. 28, 1996, pending, which is a divisional of application Ser. No. 08/441,291, filed May 15, 1995, now issued as U.S. Pat. No. 5,739,029, which is a divisional of application Ser. No. 08/071,355, filed Jun. 3, 1993, now issued as U.S. Pat. No. 5,482,835, which is a continuation of application Ser. No. 07/581,714, filed Sep. 13, 1990, now abandoned.

[0002] This invention was made with government support under NIH grants HL16037 and GM21841. The government may have certain rights to this invention.

FIELD OF THE INVENTION

[0003] This invention relates to yeast cells expressing heterologous G protein coupled receptors, vectors useful for making such cells, and methods of using the same.

BACKGROUND OF THE INVENTION

[0004] The actions of many extracellular signals (for example, neurotransmitters, hormones, odorants, light) are mediated by receptors with seven transmembrane domains (G protein coupled receptors) and heterotrimeric guanine nucleotide-binding regulatory proteins (G proteins). See H. Dohlman, M. Caron, and R. Lefkowitz, Biochemistry 26, 2657 (1987); L. Stryer and H. Bourne, Ann. Rev. Cell Biol. 2, 391 (1988). Such G protein-mediated signaling systems have been identified in organisms as divergent as yeast and man. See H. Dohlman et al., supra; L. Stryer and H. Bourne, supra; K. Blumer and J. Thorner, Annu. Rev. Physiol. (in press). The β2-adrenergic receptor (βAR) is the prototype of the seven-transmembrane-segment class of ligand binding receptors in mammalian cells. In response to epinephrine or norepinephrine, βAR activates a G protein, G_(α), which in turn stimulates adenylate cyclase and cyclic adenosine monophosphate production in the cell. See H. Dohlman et al., supra; L. Stryer and H. Bourne, supra. G protein-coupled pheromone receptors in yeast control a developmental program that culminates in mating (fusion) of a and α haploid cell types to form the a/α diploid. See K. Blumer and J. Thorner, supra; I. Herskowitz, Microbiol. Rev. 52, 536 (1988).

[0005] The present invention is based on our continued research into the expression of heterologous G protein coupled receptors in yeast.

SUMMARY OF THE INVENTION

[0006] A first aspect of the present invention is a transformed yeast cell containing a first heterologous DNA sequence which codes for a mammalian G protein coupled receptor and a second heterologous DNA sequence which codes for a mammalian G protein α subunit (mammalian G_(α)). The first and second heterologous DNA sequences are capable of expression in the cell, but the cell is incapable of expressing an endogenous G protein α-subunit (yeast G_(α)). The cell optionally contains a third heterologous DNA sequence, with the third heterologous DNA sequence comprising a pheromone-responsive promotor and an indicator gene positioned downstream from the pheromone-responsive promoter and operatively associated therewith.

[0007] A second aspect of the present invention is a method of testing a compound for the ability to affect the rate of dissociation of G_(α) from G_(βτ) in a cell. The method comprises: providing a transformed yeast cell as described above; contacting the compound to the cell; and then detecting the rate of dissociation of G_(α) from G_(βτ) in the cell. The cells may be provided in an aqueous solution, and the contacting step carried out by adding the compound to the aqueous solution.

[0008] A third aspect of the present invention is a DNA expression vector capable of expressing a transmembrane protein into the cell membrane of yeast cells. The vector contains a first segment comprising at least a fragment of the extreme amino-terminal coding sequence of a yeast G protein coupled receptor. A second segment is positioned downstream from the first segment (and in correct reading frame therewith), with the second segment comprising a DNA sequence encoding a heterologous G protein coupled receptor.

[0009] A fourth aspect of the present invention is a yeast cell transformed by a vector as described above.

BRIEF DESCRIPTION OF THE DRAWINGS

[0010]FIG. 1 illustrates the construction of the yeast human β2 Adrenergic Receptor expression plasmid, pYβAR2.

[0011]FIG. 2 illustrates hβAR ligand binding to membranes from pYβAR2-transformed yeast cells.

[0012]FIG. 3 shows a comparison of β-adrenergic agonist effects on pheromone-inducible gene activity. α-MF, 10 μM α-mating factor; (−) ISO, 50 μM (−) isoproterenol; (−) ALP, 50 μM (−) alprenolol; (+) ISO, 100 μM (+) isoproterenol.

DETAILED DESCRIPTION OF THE INVENTION

[0013] Nucleotide bases are abbreviated herein as follows: A = Adenine G = Guanine C = Cytosine T = Thymine

[0014] Amino acid residues are abbreviated herein to either three letters or a single letter as follows: Ala; A = Alanine Leu; L = Leucine Arg; R = Arginine Lys; K = Lysine Asn; N = Asparagine Met; M = Methionine Asp; D = Aspartic acid Phe; F = Phenylalanine Cys; C = Cysteine Pro; P = Proline Gln; Q = Glutamine Ser; S = Serine Glu; E = Glutamic acid Thr; T = Threonine Gly; G = Glycine Trp; W = Tryptophan His; H = Histidine Tyr; Y = Tyrosine Ile; I = Isoleucine Val; V = Valine

[0015] The term “mammalian” as used herein refers to any mammalian species (e.g., human, mouse, rat, and monkey).

[0016] The term “heterologous” is used herein with respect to yeast, and hence refers to!DNA sequences, proteins, and other materials originating from organisms other than yeast (e.g., mammalian, avian, amphibian), or combinations thereof not naturally found in yeast.

[0017] The terms “upstream” and “downstream” are used herein to refer to the direction of transcription and translation, with a sequence being transcribed or translated prior to another sequence being referred to as “upstream” of the latter.

[0018] G proteins are comprised of three subunits: a guanyl-nucleotide binding α subunit; a β subunit; and a τ subunit. G proteins cycle between two forms, depending on whether GDP or GTP is bound thereto. When GDP is bound the G protein exists as an inactive heterotrimer, the G_(αβτ)complex. When GTP is bound the α subunit dissociates, leaving a Gβτ complex. Importantly, when a G_(αβτ) complex operatively associates with an activated G protein coupled receptor in a cell membrane, the rate of exchange of GTP for bound GDP is increased and, hence, the rate of dissociation of the bound the a subunit from the G_(βτ) complex increases. This fundamental scheme of events forms the basis for a multiplicity of different cell signaling phenomena. See generally Stryer and Bourne, supra.

[0019] Any mammalian G protein coupled receptor, and the DNA sequences encoding these receptors, may be employed in practicing the present invention. Examples of such receptors include, but are not limited to, dopamine receptors, muscarinic cholinergic receptors, α-adrenergic receptors, β-adrenergic receptors, opiate receptors, cannabinoid receptors, and serotonin receptors. The term receptor as used herein is intended to encompass subtypes of the named receptors, and mutants and homologs thereof, along with the DNA sequences encoding the same.

[0020] The human D₁ dopamine receptor cDNA is reported in A. Dearry et al., Nature 347, 72-76 (1990).

[0021] The rat D₂ dopamine receptor cDNA is reported in J. Bunzow et al., Nature 336, 783-787 (1988); see also O. Civelli, et al., PCT Appln. WO 90/05780 (all references cited herein are to be incorporated herein by reference).

[0022] Muscarinic cholinergic receptors (various subtypes) are disclosed in E. Peralta et al., Nature 343, 434 (1988) and K. Fukuda et al., Nature 327, 623 (1987).

[0023] Various subtypes of α₂-adrenergic receptors are disclosed in J. Regan et al., Proc. Natl. Acad. Sci. USA 85, 6301 (1988) and in R. Lefkowitz and M. Caron, J. Biol. Chem. 263, 4993 (1988).

[0024] Serotonin receptors (various subtypes) are disclosed in S. Peroutka, Ann. Rev. Neurosci. 11, 45 (1988).

[0025] A cannabinoid receptor is disclosed in L. Matsuda et al., Nature 346, 561 (1990).

[0026] Any DNA sequence which codes for a mammalian G α subunit (G_(α)) may be used to practice the present invention. Examples of mammalian G α subunits include G_(s) α subunits, G_(i) α subunits, G_(o) α subunits, G_(z) α subunits, and transducin α subunits. See generally Stryer and Bourne, supra. G proteins and subunits useful for practicing the present invention include subtypes, and mutants and homologs thereof, along with the DNA sequences encoding the same.

[0027] Heterologous DNA sequences are expressed in a host by means of an expression vector. An expression vector is a replicable DNA construct in which a DNA sequence encoding the heterologous DNA sequence is operably linked to suitable control sequences capable of effecting the expression of a protein or protein subunit coded for by the heterologous DNA sequence in the intended host. Generally, control sequences include a transcriptional promoter, an optional operator sequence to control transcription, a sequence encoding suitable mRNA ribosomal binding sites, and (optionally) sequences which control the termination of transcription and translation.

[0028] Vectors useful for practicing the present invention include plasmids, viruses (including phage), and integratable DNA fragments (i.e., fragments integratable into the host genome by homologous recombination). The vector may replicate and function independently of the host genome, as in the case of a plasmid, or may integrate into the genome itself, as in the case of an integratable DNA fragment. Suitable vectors will contain replicon and control sequences which are derived from species compatible with the intended expression host. For example, a promoter operable in a host cell is one which binds the RNA polymerase of that cell, and a ribosomal binding site operable in a host cell is one which binds the endogenous ribosomes of that cell.

[0029] DNA regions are operably associated when they are functionally related to each other. For example: a promoter is operably linked to a coding sequence if it controls the transcription of the sequence; a ribosome binding site is operably linked to a coding sequence if it is positioned so as to permit translation. Generally, operably linked means contiguous and, in the case of leader sequences, contiguous and in reading phase.

[0030] Transformed host cells of the present invention are cells which have been transformed or transfected with the vectors constructed using recombinant DNA techniques and express the protein or protein subunit coded for by the heterologous DNA sequences. In general, the host cells are incapable of expressing an endogenous G protein α-subunit (yeast G_(α)). The host cells do, however, express a complex of the G protein β subunit and the G protein τ subunit (G_(βτ)). The host cells may express endogenous G_(βτ), or may optionally be engineered to express heterologous G_(βτ) (e.g., mammalian) in the same manner as they are engineered to express heterologous G_(α).

[0031] A variety of yeast cultures, and suitable expression vectors for transforming yeast cells, are known. See, e.g., U.S. Pat. No. 4,745,057; U.S. Pat. No. 4,797,359; U.S. Pat. No. 4,615,974; U.S. Pat. No. 4,880,734; U.S. Pat. No. 4,711,844; and U.S. Pat. No. 4,865,989. Saccharomyces cerevisiae is the most commonly used among the yeast, although a number of other strains are commonly available. See, e.g., U.S. Pat. No. 4,806,472 (Kluveromyces lactis and expression vectors therefor); U.S. Pat. No. 4,855,231 (Pichia pastoris and expression vectors therefor). Yeast vectors may contain an origin of replication from the 2 micron yeast plasmid or an autonomously replicating sequence (ARS), a promoter, DNA encoding the heterologous DNA sequences, sequences for polyadenylation and transcription termination, and a selection gene. An exemplary plasmid is YRp7, (Stinchcomb et al., Nature 282, 39 (1979); Kingsman et al., Gene 7, 141 (1979); Tschemper et al., Gene 10, 157 (1980)). This plasmid contains the trpl gene, which provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example ATCC No. 44076 or PEP4-1 (Jones, Genetics 85, 12 (1977)). The presence of the trpl lesion in the yeast host cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan.

[0032] Suitable promoting sequences in yeast vectors include the promoters for metallothionein, 3-phosphoglycerate kinase (Hitzeman et al., J. Biol. Chem. 255, 2073 (1980) or other glycolytic enzymes (Hess et al., J. Adv. Enzyme Reg. 7, 149 (1968); and Holland et al., Biochemistry 17, 4900 (1978)), such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase. Suitable vectors and promoters for use in yeast expression are further described in R. Hitzeman et al., EPO Publn. No. 73,657. Other promoters, which have the additional advantage of transcription controlled by growth conditions, are the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, and the aforementioned metallothionein and glyceraldehyde-3-phosphate dehydrogenase, as well as enzymes responsible for maltose and galactose utilization.

[0033] In constructing suitable expression plasmids, the termination sequences associated with these genes may also be ligated into the expression vector 3′ of the heterologous coding sequences to provide polyadenylation and termination of the mRNA.

[0034] A novel DNA expression vector described herein which is particularly useful for carrying out the present invention contains a first segment comprising at least a fragment of the extreme amino-terminal coding sequence of a yeast G protein coupled receptor and a second segment downstream from said first segment and in correct reading frame therewith, the second segment comprising a DNA sequence encoding a heterologous G protein coupled receptor (e.g., a mammalian G protein coupled receptor). In a preferred embodiment, this vector comprises a plasmid. In constructing such a vector, a fragment of the extreme amino-terminal coding sequence of the heterologous G protein coupled receptor may be deleted. The first and second segments are operatively associated with a promoter, such as the GAL1 promoter, which is operative in a yeast cell. Coding sequences for yeast G protein coupled receptors which may be used in constructing such vectors are exemplified by the gene sequences encoding yeast phereomone receptors (e.g., the STE2 gene, which encodes the α-factor receptor, and the STE3 gene, which encodes the α-factor receptor). The levels of expression obtained from these novel vectors are enhanced if at least a fragment of the 5′-untranslated region of a yeast G protein coupled receptor gene (e.g., a yeast pheromone receptor gene; see above) is positioned upstream from the first segment and operatively associated therewith.

[0035] Any of a variety of means for detecting the dissociation of G_(α) from G_(βτ) can be used in connection with the present invention. The cells could be disrupted and the proportion of these subunits and complexes determined physically (i.e., by chromatography). The cells could be disrupted and the quantity of G_(α) present assayed directly by assaying for the enzymatic activity possessed by G_(α) in isolation (i.e., the ability to hydrolyze GTP to GDP). Since whether GTP or GDP is bound to the G protein depends on whether the G protein exists as a G_(βτ) or G_(αβτ) complex, dissociation can be probed with radiolabelled GTP. As explained below, morphological changes in the cells can be observed. A particularly convenient method, however, is to provide in the cell a third heterologous DNA sequence, wherein the third heterologous DNA sequence comprises a pheromone-responsive promotor and an indicator gene positioned downstream from the pheromone-responsive promoter and operatively associated therewith. This sequence can be inserted with a vector, as described in detail herein. With such a sequence in place, the detecting step can be carried out by monitoring the expression of the indicator gene in the cell. Any of a variety of pheromone responsive promoters could be used, examples being the BAR1 gene promoter and the FUS1 gene promoter. Likewise, any of a broad variety of indicator genes could be used, with examples including the HIS3 gene and the LacZ gene.

[0036] As noted above, transformed host cells of the present invention express the protein or protein subunit coded for by the heterologous DNA sequence. When expressed, the G protein coupled receptor is located in the host cell membrane (i.e., physically positioned therein in proper orientation for both the stereospecific binding of ligands on the extracellular side of the cell membrane and for functional interaction with G proteins on the cytoplasmic side of the cell membrane).

[0037] The ability to control the yeast pheromone response pathway by expression of a heterologous adrenergic receptor and its cognate G protein α-subunit has the potential to facilitate structural and functional characterization of mammalian G protein-coupled receptors. By scoring for responses such as growth arrest or β-galactosidase induction, the functional properties of mutant receptors can now be rapidly tested. Similarly, as additional genes for putative G protein-coupled receptors are isolated, numerous ligands can be screened to identify those with activity toward previously unidentified receptors. See F. Libert et al., Science 244, 569 (1989); M. S. Chee et al., Nature 344, 774 (1990). Moreover, as additional genes coding for putative G protein α-subunits are isolated, they can be expressed in cells of the present invention and screened with a variety of G protein coupled receptors and ligands to characterize these subunits. These cells can also be used to screen for compounds which affect receptor-G protein interactions.

[0038] Cells of the present invention can be deposited in the wells of microtiter plates in known, predetermined quantities to provide standardized kits useful for screening compounds in accordance with the various screening procedures described above.

[0039] The following Examples are provided to further illustrate various aspects of the present invention. They are not to be construed as limiting the invention.

EXAMPLE 1 Construction of the Human β2-Adrenergic Expression Vector pYβAR2 and Expression in Yeast

[0040] To attain high level expression of the human β2-adrenergic receptor (hβAR) in yeast, a modified hβAR gene was placed under the control of the GAL1 promoter in the multicopy vector, YEp24 (pYβAR2).

[0041]FIG. 1 illustrates the construction of yeast expression plasmid pYβAR2. In pYβAR2, expression of the hβAR sequence is under the control of the GAL1 promoter. FIG. 1A shows the 5′-untranslated region and the first 63 basepairs (bp) of coding sequence of the hβAR gene in PTZNAR, B. O'Dowd et al., J. biol. Chem. 263, 15985 (1988), which was removed by Aat II cleavage and replaced with a synthetic oligonucleotide corresponding to 11 bp of noncoding and 42 bp of coding sequence from the STE.2 gene. See N. Nakayama et al., EMBO J. 4, 2643 (1985); A. Burkholder and L. Hartwell, Nucleic Acids Res. 13, 8463 (1985). The resulting plasmid, pTZYNAR, contains the modified hβAR gene flanked by Hind III sites in noncoding sequences. The Hind III-Hind III fragment was isolated from pTZYNAR and inserted into pAAH5 such that the 3′-untranslated sequence of the modified hβAR gene was followed by 450 bp containing termination sequences from the yeast ADH1 gene. See G. Ammerer, Methods. Enzymol. 1, 192 (1983).

[0042] As illustrated in FIG. 1B, pyβ13AR2 was constructed by inserting the Bam HI-Bam HI fragment containing hβAR and ADJ1 sequences into YEpG24. E. Wyckoff and T. Hsieh, Proc. Natl. Acad. Sci. U.S.A. 85, 6272 (1988). Where maximum expression was sought, cells were cotransformed with plasmid pMTL9 (from Dr. S. Johnston) containing LAC9, a homolog of the S. cerevisiae GAL4 transactivator protein required for GAL1-regulated transcription. J. Salmeron et al., Mol. Cell. Biol. 9, 2950 (1989). Cells grown to late exponential phase were induced in medium containing 3% galactose, supplemented with about 10 μM alprenolol, and grown for an additional 36 hours. Standard methods for the maintenance of cells were used. See F. Sherman et al., Methods in Yeast Genetics (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1986).

[0043] Maximal expression required (i) expression of a transcriptional transactivator protein (LAC9), (ii) replacement of the 5′ untranslated and extreme NH₂-terminal coding sequence of the hβAR gene with the corresponding region of the yeast STE2 (α-factor receptor) gene, (iii) induction with galactose when cell growth reached late exponential phase, and, (iv) inclusion of an adrenergic ligand in the growth medium during induction.

[0044] The plasmid pYβAR2 was deposited in accordance with the provisions of the Budapest Treaty at the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Md. 20852 USA, on Sep. 11, 1990, and has been assigned ATCC Accession No. 40891.

EXAMPLE 2 Binding Affinity of hβAR Ligands in Yeast Transformed with pYβAR2

[0045] A primary function of cell surface receptors is to recognize only appropriate ligands among other extracellular stimuli. Accordingly, ligand binding affinities were determined to establish the functional integrity of hβAR expressed in yeast. As discussed in detail below, an antagonist, ¹²⁵I-labeled cyanopindolol (¹²⁵I-CYP), bound in a saturable manner and with high affinity to membranes prepared from pYβAR2-transformed yeast cells. By displacement of ¹²⁵I-CYP with a series of agonists, the order of potency and stereospecificity expected for hβAR was observed.

[0046] SC261 cells (MATa ura3-52 trpl leu2 prb1-1122 pep4-3 prcl-407) (from Dr. S. Johnston) harboring pYβAR2 (URA3) and pMTL9 (LEU2) were grown in minimal glucose-free selective media to late log phase (OD₆₀₀=5.0), and then induced with the addition of 3% galactose and 40 μM alprenolol. After 36 hours, cells were harvested and spheroplasts were prepared as described. See E. Wyckoff and T. Hsieh, Proc. Natl. Acad. Sci. U.S.A. 85, 6272 (1988). Briefly, the spheroplasts were resuspended in 50 mM Tris-HCl pH 7.4, 5 mM EDTA and were lysed by vortex mixing with glass beads for three one-min periods at 4° C. Crude membranes were prepared from the lysates and binding assays with ¹²⁵I-CYP were performed by methods described previously. See H. Dohlman et al., Biochemistry 29, 2335 (1990).

[0047]FIG. 2 illustrates hβAR ligand binding to membranes from pYβAR2-transformed yeast cells. (A) B_(max) (maximum ligand bound) and K_(d) (ligand dissociation constant) values were determined by varying 125_(I)-CYP concentrations (5-400 pM). Specific binding was defined as the amount of total binding (circles) minus nonspecific binding measured in the presence of 10 μM (−) alprenolol (squares). A K_(d) of 93 μM for 125_(I)-CYP binding was obtained and used to calculate agonist affinities (below). (B) Displacement of 18 pM 125_(I)-CYP with various concentrations of agonists was used to determine apparent low affinity K_(i) values (non G protein coupled, determined in the presence of 50 μM GTP) for receptor binding, squares; (−) isoproterenol, circles; (−) epinephrine, downward-pointing triangles; (+) isoproterenol, upward pointing triangles; (−) norepinephrine. COMPARATIVE EXAMPLE A

Ligand Binding Affinity for hβAR Expressed in Yeast and Mammalian Cells

[0048] The binding data of FIGS. 2(A) and (B) were analyzed by nonlinear least squares regression, see A. DeLean et al., Mol. Pharmacol. 21, (1982), and are presented in Table I. Values given are averages of measurements in triplicate, and are representative of 2-3 experiments. Binding affinities in yeast were nearly identical to those observed previously for hβAR expressed in mammalian cells. TABLE 1 Comparison of ligand Binding Parameters for High Level Expression of Human β-Adrenergic Receptor in Yeast and COS-7 Cells* Yeast Monkey SC261 COS-7 (pY, βAR2, pMTL9) (pBC12: β,BAR) 125₁-CYP: ¹K_(d) 0.093 nM ± 0.013 0.110 nM ± 0.009 ²B_(max) 115 pmol/mg 24 pmol/mg ³K₁(M): (−) isoproterenol 103 ± 26  130 ± 15  (+) isoproterenol 3670 ± 420  4000 ± 184  (−) epinephrine 664 ± 123 360 ± 30  (−) norepinephrine 6000 ± 1383 5800 ± 373 

EXAMPLE 3 Agonist-Dependent Activation of Mating Signal Transduction in Yeast Expressing hβAR

[0049] A second major function of a receptor is agonist-dependent regulation of downstream components in the signal transduction pathway. Because the pheromone-responsive effector in yeast is not known, indirect biological assays are the most useful indicators of receptor functionality. See K. Blumer and J. Thorner, Annu. Rev. Physiol. in press; I. Herskowitz, Microbiol. Rev. 52, 536 (1988). In yeast cells expressing high concentrations of hβAR, no agonist-dependent activation of the mating signal transduction pathway could be detected by any of the typical in vivo assays; for example, imposition of G1 arrest, induction of gene expression, alteration of morphology (so-called “shmoo” formation) or stimulation of mating. A likely explanation for the absence of responsiveness is that hβAR was unable to couple with the endogenous yeast G protein.

EXAMPLE 4 Coexpression of hβAR and Mammalian G_(s) α-Subunit in Yeast

[0050] Expression of a mammalian G_(s) α-subunit can correct the growth defect in yeast cells lacking the corresponding endogenous protein encoded by the GPA1 gene. See C. Dietzel and J. Kurjan, Cell 50, 1001 (1987). Moreover, specificity of receptor coupling in mammalian cells is conferred by the α-subunit of G proteins. See L. Stryer and H. Bourne, Annu. Rev. Cell Biol. 2, 391 (1988). Thus, coexpression of hβAR and a mammalian G_(s) α-subunit (GSα) in yeast was attempted to render the yeast responsive to adrenergic ligands. Accordingly, a cDNA encoding rat G_(s)α under the control of the copper-inducible CUP1 promoter was introduced on a second plasmid, pYSK136Gαs. See C. Dietzel and J. Kurjan, Cell 50, 1001 (1987). In yeast (NNY19) coexpressing hβAR and rat G_(s)α, but containing wild-type GPA1, no adrenergic agonist-induced shmoo formation, a characteristic morphological change of yeast in response to mating pheromone, was observed.

EXAMPLE 5 Coexpression of hβAR and Mammalian G_(s)α-Subunit in Yeast Lacking an Endogenous G Protein α-Subunit

[0051] To prevent interference by the endogenous yeast G protein α-subunit, qpal mutant cells (strain 8c) were used.

[0052] Yeast strain 8c (MATa ura3 leu2 his3 trpl gpal::Hl53), I. Miyajima et al., Cell 50, 1011 (1987), carrying plasmids pYSK136Gαs (TR1), C. Dietzel and J. Kurjan, Cell 50, 1001 (1987), pMTL9 (LEU2), J. Salmeron et al., Mol. Cell. Biol. 9, 2950 (1989), and pYβAR2 (URA3) was maintained on glucose-free minimal selective plates containing 3% glycerol, 2% lactic acid, 50 μM CuSO₄ and 3% galactose. Colonies were transferred to similar plates containing 0.5 mM ascorbic acid and the indicated adrenergic ligand(s). After 16-20 hours at 30° C., the colonies were transferred to similar liquid media at a density of 10⁶-10⁷ cells/ml and examined by phase contrast microscopy.

[0053] Morphologies of yeast cells cotransformed with pYβAR2, pMTL9, and pYSK136Gαs were examined after incubation with (A) no adrenergic agent; (B) 100 μM (−) isoproterenol; (C) 100 μM (−) isoproterenol and 50 μM (−) alprenolol; and (D) 100 μM (+) isoproterenol. Results showed that treatment of 8c cells coexpressing hβAR and rat G_(s)α with the β-adrenergic agonist isoproterenol indeed induced shmoo formation, and that this effect was blocked by the specific antagonist alprenolol.

EXAMPLE 6 Coexpression of hβAR and Mammalian G_(s)α-Subunit in Yeast Containing a β-Galactosidase Signal Sequence

[0054] The isoproterenol-induced morphological response of 8c cells coexpressing hβAR and rat G_(s)α suggested that these components can couple to each other and to downstream components of the pheromone response pathway in yeast lacking the endogenous G α-subunit. To confirm that the pheromone signaling pathway was activated by hβAR and rat G_(s)α, agonist induction of the pheromone-responsive FUSl gene promoter was measured in a strain of yeast derived from 8c cells (8cl) in which a FUSl-lacZ gene fusion had been stably integrated into the genome. See S. Nomoto et al., EMBO J. 9, 691 (1990).

[0055] Strains 8c (FIG. 3, legend) and NNYl9 (MATa ura3 leu2 his3 trpl lys2 FUS1-LacZ::LEU2) were modified by integrative transformation with YIpFUS102 (LEU2), S. Nomoto et al., supra, and designated 8cl and NNYl9, respectively. These strains were transformed with pYβAR2 and pYSK136Gαs and maintained on minimal selective plates containing glucose and 50 μM CuSO₄. Colonies were inoculated into minimal selective media (3% glycerol, 2% lactic acid, 50 μM CuSo₄), grown to early log phase (OD₆₀₀=1.0), and induced for 12 hours by addition of 3% galactose. Cells were washed and resuspended in induction media (OD₆₀₀=5.0) containing 0.5 mM ascorbic acid and the indicated ligands. After a 4 hour incubation at 30° C., cells were harvested, resuspended into 1 ml of Z-buffer, see J. Miller, Experiments in Molecular Genetics (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1972), supplemented with 0.0075% SDS, and β-galactosidase activities were determined in 3-4 independent experiments as described previously. See J. Miller, supra.

[0056]FIG. 3 shows a comparison of β-adrenergic agonist effects on pheromone-inducible gene activity. α-MF, 10 μM α-mating factor; (−) ISO, 50 μM (−) isoproterenol; (−) ALP, 50 μM (−) alprenolol; (+) ISO, 100 μM (+) isoproterenol. In 8cl (gpal) cells coexpressing hβAR and rat G_(s)α, a dramatic isoproterenol-stimulated induction of β-galactosidase activity was observed. Agonist stimulation was stereoselective and was blocked by addition of a specific antagonist. Agonist responsiveness was dependent on expression of both hβAR and rat G_(s)α, and required a strain in which the endogenous G protein α-subunit was disrupted. The final β-galactosidase activity achieved in response to isoproterenol in transformed 8cl cells was comparable to that induced by α-factor in nontransformed cells that express GPA1 (NNY19), although basal β-galactosidase activity in NNY19 cells was considerably lower than in 8cl cells. Taken together, our results indicated that coexpression of hβAR and rat G_(s)α was sufficient to place under catecholamine control key aspects of the mating signal transduction pathway in yeast. However, the adrenergic agonist did not stimulate mating in either 8c cells or NNY19 cells coexpressing hβAR and rat G_(s)α, in agreement with recent observations that yeast pheromone receptors, in addition to binding pheromones, participate in other recognition events required for mating. See A. Bender and G. Sprague, Genetics 121, 463 (1989).

[0057] hβAR stimulates adenylate cyclase in animal cells via the action of the α-subunit of its G protein. In contrast, mating factor receptors in yeast trigger their effector via the action of the βτ subunits. M. Whiteway et al., Cell 56, 476 (1989). Our present results indicate that activation of hβAR in yeast leads to dissociation of mammalian G_(s)α from yeast βτ, and it is the βτ subunits that presumably elicit the response.

[0058] The foregoing examples are illustrative of the present invention, and are not to be construed as limiting thereof. The invention is defined by the following claims, with equivalents of the claims to be included therein. 

That which is claimed is:
 1. A transformed yeast cell containing a first heterologous DNA sequence which codes for a mammalian G protein coupled receptor and a second heterologous DNA sequence which codes for a mammalian G protein a subunit (mammalian G_(α)), wherein said first and second heterologous DNA sequences are capable of expression in said cell, and wherein said cell is incapable of expressing an endogenous G protein α-subunit (yeast G_(α)).
 2. A transformed yeast cell according to claim 1, wherein said first heterologous DNA sequence is carried by a plasmid.
 3. A transformed yeast cell according to claim 1, wherein said second heterologous DNA sequence is carried by a plasmid.
 4. A transformed yeast cell according to claim 1, wherein said mammalian G protein α subunit is selected from the group consisting of G_(s) α subunits, G_(i) α subunits, G_(o) α subunits, G_(z) α subunits, and transducin α subunits.
 5. A transformed yeast cell according to claim 1 which expresses a complex of the G protein β subunit and the G protein τ subunit (G_(βτ)).
 6. A transformed yeast cell according to claim 5 which expresses endogenous G_(βτ).
 7. A transformed yeast cell according to claim 1, wherein said first heterologous DNA sequence codes for a mammalian G protein-coupled receptor selected from the group consisting of dopamine receptors, muscarinic cholinergic receptors, α-adrenergic receptors, β-adrenergic receptors, opiate receptors, cannabinoid receptors, and serotonin receptors.
 8. A transformed yeast cell according to claim 1 further comprising a third heterologous DNA sequence, wherein said third heterologous DNA sequence comprises a pheromone-responsive promotor and an indicator gene positioned downstream from said pheromone-responsive promoter and operatively associated therewith.
 9. A transformed yeast cell according to claim 8, wherein said pheromone responsive promoter is selected from the group consisting of the BAR1 gene promoter and the FUS1 gene promoter, and wherein said indicator gene is selected from the group consisting of the HIS3 gene and the LacZ gene.
 10. A method of testing a compound for the ability to affect the rate of dissociation of G_(α) from G_(βτ) in a cell, comprising: providing a transformed yeast cell containing a first heterologous DNA sequence which codes for a mammalian G protein coupled receptor and a second heterologous DNA sequence which codes for a mammalian G_(α), wherein said first and second heterologous DNA sequences are capable of expression in said cell, wherein said cell is incapable of expressing endogenous G_(α), and wherein said cell expresses G_(βτ); contacting said compound to said cell; and detecting the rate of dissociation of G_(a) from G_(βτ) in said cell.
 11. A method according to claim 10, wherein said yeast cells are provided in an aqueous solution and said contacting step is carried out by adding said compound to said aqueous solution.
 12. A method according to claim 10, wherein said mammalian G protein a subunit is selected from the group consisting of G_(s) α subunits, G_(i) α subunits, G_(o) α subunits, G_(z) α subunits, and transducin α subunits.
 13. A method according to claim 10, wherein said yeast cell expresses endogenous G_(βτ).
 14. A method according to claim 10, wherein said first heterologous DNA sequence codes for a mammalian G protein-coupled receptor selected from the group consisting of dopamine receptors, muscarinic cholinergic receptors, α-adrenergic receptors, β-adrenergic receptors, opiate receptors, cannabinoid receptors, and serotonin receptors.
 15. A method according to claim 10, said yeast cell further comprising a third heterologous DNA sequence, wherein said third heterologous DNA sequence comprises a pheromone-responsive promotor and an indicator gene positioned downstream from said pheromone-responsive promoter and operatively associated therewith; and wherein said detecting step is carried out by monitoring the expression of said indicator gene in said cell.
 16. A DNA expression vector capable of expressing a transmembrane protein into the cell membrane of yeast cells, comprising: a first segment comprising at least a fragment of the extreme amino-terminal coding sequence of a yeast G protein coupled receptor; and a second segment downstream from said first segment and in correct reading frame therewith, said second segment comprising a DNA sequence encoding a heterologous G protein coupled receptor.
 17. A DNA expression vector according to claim 16, wherein a fragment of the extreme amino-terminal coding sequence of said heterologous G protein coupled receptor is absent.
 18. A DNA expression vector according to claim 16, wherein said first and second segments are operatively associated with a promoter operative in a yeast cell.
 19. A DNA expression vector according to claim 18, wherein said promoter is the GAL1 promoter.
 20. A DNA expression vector according to claim 16, wherein said first segment comprises at least a fragment of the extreme amino-terminal coding sequence of a yeast phereomone receptor.
 21. A DNA expression vector according to claim 16, wherein said first segment comprises at least a fragment of the extreme amino-terminal coding sequence of a yeast phereomone receptor selected from the group consisting of the STE2 gene and the STE3 gene.
 22. A DNA expression vector according to claim 16, further comprising at least a fragment of the 5′-untranslated region of a yeast G protein coupled receptor gene positioned upstream from said first segment and operatively associated therewith.
 23. A DNA expression vector according to claim 16, further comprising at least a fragment of the 5′-untranslated region of a yeast phereomone receptor gene positioned upstream from said first segment and operatively associated therewith.
 24. A DNA expression vector according to claim 23, wherein said yeast pheromone receptor gene is selected from the group consisting of the STE2 gene and the STE3 gene.
 25. A DNA expression vector according to claim 16, said vector comprising a plasmid.
 26. A DNA expression vector according to claim 16, said second segment comprising a DNA sequence encoding a mammalian G protein coupled receptor.
 27. A DNA expression vector according to claim 16, said second segment comprising a DNA sequence encoding a mammalian G protein-coupled receptor selected from the group consisting of dopamine receptors, muscarinic cholinergic receptors, α-adrenergic receptors, β-adrenergic receptors, opiate receptors, cannabinoid receptors, and serotonin receptors.
 28. A yeast cell carrying a DNA expression vector according to claim
 16. 29. A method for screening a plurality of compounds to identify a compound that agonizes or antagonizes a G protein-coupled receptor-mediated activity by detecting intracellular transduction of a signal generated upon interaction of the compound with the G protein-coupled receptor, comprising: comparing the amount of expression of an indicator gene product in a recombinant eukaryotic cell in the presence of the compound with the amount of expression of an indicator gene product in the absence of the compound; and selecting the compound from the plurality of compounds that changes the amount of expression of the indicator gene product in the recombinant cell in the presence of the compound compared to the amount of expression of an indicator gene product in the absence of the compound whereby the compound that modulates G protein-coupled receptor mediated activity is identified, wherein: the recombinant cell contains an indicator gene construct and expresses the G protein-coupled receptor; and the indicator gene construct includes: (a) a transcriptional control element that is responsive to the intracellular signal that is generated by the interaction of the compound with the G protein-coupled receptor; and (b) an indicator gene that encodes a detectable product and that is in operative association with the transcriptional control element.
 30. The method of claim 29, wherein said compound is an agonist or antagonist of said G protein-coupled receptor.
 31. The method of claim 29, wherein the amount of expression of an indicator gene product is measured by measuring the amount of indicator gene protein that is produced.
 32. The method of claim 30, wherein said compound is an antagonist.
 33. The method of claim 30, further comprising, prior to comparing the difference in the amount of expression of the indicator gene product, contacting the recombinant cell with an agonist that activates said cell surface protein, whereby expression of said indicator gene product is induced.
 34. The method of claim 29, wherein the G protein-coupled receptor is selected from the group consisting of muscarinic receptors, adrenergic receptors, dopamine receptors, and serotonin receptors.
 35. The method of claim 29, wherein the transcriptional control element includes a promoter sequence which is responsive to the intracellular signal.
 36. A method for screening a plurality of compounds to identify a compound that agonizes or antagonizes a G protein-coupled receptor-mediated activity by detecting intracellular transduction of a signal generated upon interaction of the compound with the G protein-coupled receptor, comprising: comparing the amount of expression of an indicator gene product in a first recombinant eukaryotic cell in the presence of the compound with the amount of expression of an indication gene product in the absence of the compound, or with the amount of expression in a second recombinant cell; and selecting a compound from the plurality of compounds that changes the amount of expression of the indicator gene product in the first recombinant cell in the presence of the compound compared to the amount of expression in the absence of the compound, or compared to the amount of expression in the second recombinant cell, whereby a compound that modulates G protein-coupled receptor-mediated activity is identified wherein: the first recombinant eukaryotic cell contains an indicator gene construct and expresses the G protein-coupled receptor; the second recombinant cell is identical to the first recombinant cell, except that it does not express the G protein-coupled receptor; and the indicator gene construct includes: (a) a transcriptional control element that is responsive to the intracellular signal that is generated by the interaction of the compound with the G protein-coupled receptor; and (b) an indicator gene that encodes a detectable product and that is in operative association with the transcriptional control element.
 37. A method for identifying extracellular signals that agonize or antagonize cell surface protein-mediated activity, comprising: comparing the difference in the amount of expression of an indicator gene product in a recombinant cell in the presence of the signal with the amount of expression in the absence of said signal, or with the amount of expression in the absence of the cell surface protein, wherein: the recombinant cell contains an indicator gene construct and expresses the cell surface protein; and expression of the indicator gene product is under the control of at least one transcriptional control element whose activity is regulated by the cell surface protein.
 38. The method of claim 37, wherein said protein is a cell surface receptor.
 39. The method of claim 38, wherein said signal is an agonist or antagonist of said cell surface receptor.
 40. The method of claim 39, wherein the concentration of said agonist or antagonist is less than 100 μM.
 41. The method of claim 39, wherein the concentration of said agonist or antagonist is less than 10 μM.
 42. The method of claim 39, wherein the concentration of said agonist or antagonist is less than 1 μM.
 43. The method of claim 39, wherein the concentration of said agonist or antagonist is less than 0.1 μM.
 44. The method of claim 39, wherein the concentration of said agonist or antagonist is less than 10 nM.
 45. The method of claim 37, wherein the amount of expression is determined by measuring the amount of indicator gene product that is produced.
 46. The method of claim 39, wherein said signal is an antagonist.
 47. The method of claim 46, further comprising, prior to or simultaneously with comparing the difference in the amount of expression of the indicator gene product, contacting the recombinant cell with an agonist that activates said cell surface protein, whereby expression of said indicator gene is induced.
 48. The method of claim 37, wherein the cell surface protein is a cell surface receptor selected from: muscarinic receptors, adrenergic receptors, dopamine receptors, serotonin receptors.
 49. The method of claim 37, wherein the transcriptional control element includes a promoter sequence which is responsive to intracellular signals.
 50. A recombinant cell, comprising: DNA that encodes a heterologous cell surface protein whose activity is modulated by extracellular signals; and a reporter gene construct containing a reporter gene in operative linkage with one or more transcriptional control element that is regulated by said cell surface protein; wherein said cell surface protein is a G-protein coupled receptor.
 51. The recombinant cell of claim 50, wherein said cell surface protein is a cell surface receptor selected from: muscarinic receptors, adrenergic receptors, dopamine receptors, serotonin receptors.
 52. A method for identifying compounds that agonize or antagonize cell surface protein-mediated activity by detecting intracellular transduction of a signal generated upon interaction of the compound with a cell surface protein, comprising: comparing the amount of expression of an indicator gene product in a first recombinant cell in the presence of the compound with the amount of indicator gene product in a second recombinant cell; and selecting compounds that change the amount of expression of an indicator gene product in the first recombinant cell in the presence of the compound compared to the amount of expression in the absence of the compound, or compared to the amount of expression in the second recombinant cell, wherein: the first recombinant cell contains an indicator gene construct and expresses the cell surface protein; the second recombinant cell is identical to the first recombinant cell, except that it does not express the cell surface protein; and the indicator gene construct contains: (a) a transcriptional control element that is responsive to the intracellular signal that is generated by the interaction of an agonist with the cell surface protein; and (b) an indicator gene that encodes a detectable product and that is in operative association with the transcriptional control element.
 53. The method of claim 52, wherein the compound is an agonist or antagonist of said cell surface receptor.
 54. The method of claim 52, wherein the level of expression is determined by measuring the amount of indicator gene product that is produced.
 55. The method of claim 52, further comprising, prior to or simultaneously with comparing the difference in the amount of indicator gene product, contacting the recombinant cell with an agonist that activates said cell surface protein, whereby expression of said indicator gene product is induced.
 56. The method of claim 55, wherein said compound is an antagonist.
 57. The method of claim 47, wherein said compound is an antagonist.
 58. The method of claim 37, wherein the cell is preincubated with the extracellular signal prior to comparing the difference in expression.
 59. The method of claim 52, wherein the cell is preincubated with the compound prior to comparing the difference in expression.
 60. The method of any one of claims 37 and 52, wherein said cell surface protein is a G-protein coupled receptor.
 61. The method of claim 36, wherein said compound is an agonist or antagonist of said G protein-coupled receptor.
 62. The method of claim 36, wherein the amount of expression of an indicator gene product is measured by measuring the amount of indicator gene protein that is produced.
 63. The method of claim 36, wherein said compound is an antagonist.
 64. The method of claim 36, further comprising, prior to comparing the difference in the amount of expression of the indicator gene product, contacting the recombinant cell with an agonist that activates said cell surface protein, whereby expression of said indicator gene product is induced.
 65. The method of claim 36, wherein the G protein-coupled receptor is selected from the group consisting of muscarinic receptors, adrenergic receptors, dopamine receptors, and serotonin receptors.
 66. An assay for screening a plurality of compounds to determine if a compound so screened has cell surface receptor agonist or antagonist activity, which assay comprises (a) challenging a recombinant eukaryotic cell with the compound, wherein the cell recombinantly expresses the G protein-coupled receptor; (b) determining the level of indicator gene product recombinantly expressed in the cell; (c) comparing the amount of expression of an indicator gene product in the eukaryotic cell with the amount of expression of an indicator gene product in the eukaryotic cell in the absence of the compound, and (d) identifying a compound from the plurality of compounds that exhibits said cell surface receptor agonist or antagonist activity, wherein the indicator gene is under transcriptional control of a transcriptional control element selected to be responsive to an intracellular signal that is generated by the interaction of a compound with G protein-coupled receptor.
 67. The assay of claim 66, wherein said compound is an agonist or antagonist of said G protein-coupled receptor.
 68. The assay of claim 66, wherein the amount of expression is measured by measuring the amount of indicator gene protein that is produced.
 69. The assay of claim 67, wherein said compound is an antagonist.
 70. The assay of claim 67, further comprising, prior to comparing the difference in the amount of expression of the indicator gene product, contacting the recombinant cell with an agonist that activates said cell surface protein, whereby expression of said indicator gene product is induced.
 71. The assay of claim 66, wherein said cell surface protein is a G-protein coupled receptor.
 72. The assay of claim 66, wherein the cell surface receptor is selected from the group consisting of muscarinic receptors, adrenergic receptors, dopamine receptors, and serotonin receptors.
 73. The assay of claim 66, wherein the transcriptional control element includes a promoter sequence which is responsive to the intracellular signal.
 74. An assay for screening a plurality of compounds to determine if a compound so screened has cell surface receptor agonist or antagonist activity, which assay that comprises (a) challenging a first recombinant eukaryotic cell with a compound to be screened wherein the cell recombinantly expresses the G protein-coupled receptor, (b) determining the level of expression of an indicator gene product recombinantly expressed in the cell, (c) comparing the amount of expression of an indicator gene product in the first recombinant eukaryotic cell with the amount of expression of an indicator gene product in a second recombinant eukaryotic cell that is identical to the first eukaryotic cell except that the second cell does not express the G protein-coupled receptor, and (d) identifying a compound that exhibits said G protein-coupled receptor agonist or antagonist activity, wherein the indicator gene is under transcriptional control of a transcriptional control element selected to be responsive to an intracellular signal that is generated by the interaction of the compound with the G protein-coupled receptor.
 75. The assay of claim 74, wherein said compound is an agonist or antagonist of G protein-coupled receptor.
 76. The assay of claim 74, wherein the amount of expression is measured by measuring the amount of indicator gene protein that is produced.
 77. The assay of claim 75, wherein said compound is an antagonist.
 78. The assay of claim 75, further comprising, prior to comparing the difference in the amount of expression of the indicator gene product, contacting the recombinant cell with an agonist that activates said cell surface protein, whereby expression of said indicator gene product is induced.
 79. The assay of claim 74, wherein the G protein-coupled receptor is selected from the group consisting of muscarinic receptors, adrenergic receptors, dopamine receptors, and serotonin receptors.
 80. The assay of claim 74, wherein the transcriptional control element includes a promoter sequence which is responsive to the intracellular signal. 